Effects on enteric methane production and bacterial and archaeal communities by the addition of cashew nut shell extract or glycerol-an in vitro evaluation.

The objective of the study was to evaluate the effect of cashew nut shell extract (CNSE) and glycerol (purity >99%) on enteric methane (CH4) production and microbial communities in an automated gas in vitro system. Microbial communities from the in vitro system were compared with samples from the donor cows, in vivo. Inoculated rumen fluid was mixed with a diet with a 60:40 forage:concentrate ratio and, in total, 5 different treatments were set up: 5mg of CNSE (CNSE-L), 10mg of CNSE (CNSE-H), 15mmol of glycerol/L (glycerol-L), and 30mmol of glycerol/L (glycerol-H), and a control without feed additive. Gas samples were taken at 2, 4, 8, 24, 32, and 48h of incubation, and the CH4 concentration was measured. Samples of rumen fluid were taken for volatile fatty acid analysis and for microbial sequence analyses after 8, 24, and 48h of incubation. In vivo rumen samples from the cows were taken 2h after the morning feeding at 3 consecutive days to compare the in vitro system with in vivo conditions. The gas data and data from microbial sequence analysis (454 sequencing) were analyzed using a mixed model and principal components analysis. These analyses illustrated that CH4 production was reduced with the CNSE treatment, by 8 and 18%, respectively, for the L and H concentration. Glycerol instead increased CH4 production by 8 and 12%, respectively, for the L and H concentration. The inhibition with CNSE could be due to the observed shift in bacterial population, possibly resulting in decreased production of hydrogen or formate, the methanogenic substrates. Alternatively the response could be explained by a shift in the methanogenic community. In the glycerol treatments, no main differences in bacterial or archaeal population were detected compared with the in vivo control. Thus, the increase in CH4 production may be explained by the increase in substrate in the in vitro system. The reduced CH4 production in vitro with CNSE suggests that CNSE can be a promising inhibitor of CH4 formation in the rumen of dairy cows.

Presence of Anaplasma spp. and Their Associated Antibodies in the Swedish Goat Population.

Anaplasmosis is a tick-borne disease that has a severe impact on livestock production and welfare. The aim of this pilot study was to investigate the presence of Anaplasma spp. and associated antibodies in a subset of the Swedish goat population. In 2020, six goat herds located in different parts of Sweden were visited and whole blood and serum samples were collected. The whole blood samples (n = 40) were analysed for the presence of Anaplasma phagocytophilum, A. ovis and A. capra using quantitative and conventional polymerase chain reaction (PCR). The serum samples (n = 59) were analysed for the presence of antibodies to Anaplasma spp. using a commercial competitive enzyme-linked immunosorbent assay, and the same analysis was carried out on additional serum samples previously collected in 2018, 2019 and 2020 (n = 166). One goat (2.5%) tested positive for the presence of A. phagocytophilum genetic material, while the seropositivity rate ranged from 20 to 71%, depending on the surveyed year and area. These results indicate widespread exposure to Anaplasma spp. in the Swedish goat population. To inform future risk assessments and control efforts, further research is warranted to determine the prevalence of anaplasmosis and its impact on goat farming in Sweden.

Ixodid tick species found in northern Sweden - Data from a frontier area.

Environmental and climatic changes in northern Europe have shaped a geographical area in which new tick species may become established and introduce new tick-borne pathogens. In recent decades, ticks have expanded their latitudinal and altitudinal range limits in northern Sweden. In this study, ticks were collected in 2018 and 2019 in northern Sweden from different hosts, mainly from dogs, cats and humans. The ticks in 2018 (n = 2141, collected from 65 municipalities in 11 provinces) were identified as Ixodes ricinus (n = 2108, 98.5%), Ixodes persulcatus (n = 18, 0.8%), Ixodes trianguliceps (n = 14, 0.7%) and Hyalomma marginatum (n = 1, 0.05%). The ticks collected in 2019 (n = 519, across a smaller area than in 2018, i.e. Sweden's four northernmost provinces) were identified as I. ricinus (n = 242, 46.6%) and I. persulcatus (n = 277, 53.4%). Among those collected in 2019, the majority of I. ricinus (n = 111, 45.9%) were submitted from the province of Västerbotten, while most I. persulcatus (n = 259, 93.5%) were collected in the province of Norrbotten. This study provides updated figures on the geographical distribution of two Ixodes species in northern Sweden. The results confirmed I. ricinus to be the dominant species and that I. persulcatus has enlarged its distributional area compared with previous reports. Updated knowledge of tick distribution is fundamental for the creation of risk maps and will allow relevant advice to be provided to the general public, suggesting measures to prevent tick bites and consequently tick-borne diseases.

Dairy milk from cow and goat as a sentinel for tick-borne encephalitis virus surveillance.

Tick-borne encephalitis (TBE) is one of the most severe human tick-borne diseases in Europe. It is caused by the tick-borne encephalitis virus (TBEV), which is transmitted to humans mainly via bites of Ixodes ricinus or I. persulcatus ticks. The geographical distribution and abundance of I. ricinus is expanding in Sweden as has the number of reported human TBE cases. In addition to tick bites, alimentary TBEV infection has also been reported after consumption of unpasteurized dairy products. So far, no alimentary TBEV infection has been reported in Sweden, but knowledge about its prevalence in Swedish ruminants is scarce. In the present study, a total of 122 bulk tank milk samples and 304 individual milk samples (including 8 colostrum samples) were collected from dairy farms (n = 102) in Sweden. All samples were analysed for the presence of TBEV antibodies by ELISA test and immunoblotting. Participating farmers received a questionnaire about milk production, pasteurization, tick prophylaxis used on animals, tick-borne diseases, and TBE vaccination status. We detected specific anti-TBEV antibodies, i.e., either positive (>126 Vienna Units per ml, VIEU/ml) or borderline (63-126 VIEU/ml) in bulk tank milk from 20 of the 102 farms. Individual milk samples (including colostrum samples) from these 20 farms were therefore collected for further analysis. Our results revealed important information for detection of emerging TBE risk areas. Factors such as consumption of unpasteurized milk, limited use of tick prophylaxis on animals and a moderate coverage of human TBE vaccination, may be risk factors for alimentary TBEV infection in Sweden.

A Disease Outbreak in Beef Cattle Associated with Anaplasma and Mycoplasma Infections.

An outbreak of disease in a Swedish beef cattle herd initiated an in-depth study to investigate the presence of bacteria and viruses in the blood of clinically healthy (n = 10) and clinically diseased cattle (n = 20) using whole-genome shotgun sequencing (WGSS). The occurrence of infectious agents was also investigated in ticks found attached to healthy cattle (n = 61) and wild deer (n = 23), and in spleen samples from wild deer (n = 30) and wild boars (n = 10). Moreover, blood samples from 84 clinically healthy young stock were analysed for antibodies against Anaplasma phagocytophilum and Babesia divergens. The WGSS revealed the presence of at least three distinct Mycoplasma variants that were most closely related to Mycoplasma wenyonii. Two of these were very similar to a divergent M. wenyonii variant previously only detected in Mexico. These variants tended to be more common in the diseased cattle than in the healthy cattle but were not detected in the ticks or wild animals. The DNA of A. phagocytophilum was detected in similar proportions in diseased (33%) and healthy (40%) cattle, while 70% of the deer, 8% of ticks collected from the cattle and 19% of the ticks collected from deer were positive. Almost all the isolates from the cattle, deer and ticks belonged to Ecotype 1. Based on sequencing of the groEL-gene, most isolates of A. phagocytophilum from cattle were similar and belonged to a different cluster than the isolates from wild deer. Antibodies against A. phagocytophilum were detected in all the analysed samples. In conclusion, uncommon variants of Mycoplasma were detected, probably associated with the disease outbreak in combination with immune suppression due to granulocytic anaplasmosis. Moreover, A. phagocytophilum was found to be circulating within this cattle population, while circulation between cattle and deer occurred infrequently.

Characterization of the bacterial microbiome of Swedish ticks through 16S rRNA amplicon sequencing of whole ticks and of individual tick organs.

BACKGROUND: The composition of the microbial flora associated with ixodid ticks has been studied in several species, revealing the importance of geographical origin, developmental stage(s) and feeding status of the tick, as well as substantial differences between tissues and organs. Studying the microbiome in the correct context and scale is therefore necessary for understanding the interactions between tick-borne pathogens and other microorganisms as well as other aspects of tick biology. METHODS: In the present study the microbial flora of whole Ixodes ricinus, I. persulcatus and I. trianguliceps ticks were analyzed with 16S rRNA amplicon sequencing. Additionally, tick organs (midguts, Malpighian tubules, ovaries, salivary glands) from flat and engorged I. ricinus female ticks were examined with the same methodology. RESULTS: The most abundant bacteria belonged to the group of Proteobacteria (Cand. Midichloria mitochondrii and Cand. Lariskella). 16S amplicon sequencing of dissected tick organs provided more information on the diversity of I. ricinus-associated microbial flora, especially when organs were collected from engorged ticks. Bacterial genera significantly associated with tick feeding status as well as genera associated with the presence of tick-borne pathogens were identified. CONCLUSIONS: These results contribute to the knowledge of microbial flora associated with ixodid ticks in their northernmost distribution limit in Europe and opens new perspectives for other investigations on the function of these bacteria, including those using other approaches like in vitro cultivation and in vitro models.

Infectious keratoconjunctivitis in semi-domesticated reindeer (Rangifer tarandus tarandus): a questionnaire-based study among reindeer herders in Norway and Sweden.

BACKGROUND: The effects of climate change, loss of pastureland to other land usage and presence of large carnivores are the main reasons for the increase in supplementary feeding of semi-domesticated reindeer (Rangifer tarandus tarandus) in Fennoscandia over the last decades. Feeding might expose reindeer to stress and increased animal-to-animal contact, leading to an increased risk of infectious disease transmission, such as infectious keratoconjunctivitis (IKC). As it can develop rapidly and be very painful, IKC is described as an important animal welfare concern and a potential source of economic loss. The aim of this study was to investigate the current presence of IKC and potential associations between IKC and supplementary feeding through an online questionnaire survey, distributed among reindeer herders in Norway and Sweden in 2021. RESULTS: Seventy-six reindeer herders (33 from Norway and 43 from Sweden) responded to the questionnaire, representing 6% and 4% of the registered reindeer herding groups in Norway and Sweden, respectively. Infectious keratoconjunctivitis was common, with 54 (71%) of the 76 herders that responded having observed clinical signs during the past 10 years. These signs were mainly observed as increased lacrimation, causing "wet cheeks", but also as keratitis and conjunctivitis. Autumn and winter were the seasons in which IKC was observed most. The herders reported several measures, such as slaughter and isolation of affected reindeer, to counteract the spread of disease. The herding year 2019/2020 was associated with reports of outbreaks of IKC in herds as well as being the herding year where most herders (80%) had performed supplementary feeding. A significant association was found between IKC and feeding performed in an enclosure (odds ratio = 15.20), while feeding on free-range areas had a non-significant, negative, relationship with the appearance of IKC outbreaks (odds ratio = 0.29). Finally, there was a trend in the data suggesting that IKC affected calves especially. CONCLUSIONS: Infectious keratoconjunctivitis is a common disease, mainly observed in winter and autumn. It usually has mild to moderately severe clinical signs. Our results imply that IKC is associated with stress and feeding situations and that calves might be more susceptible than adults, however, this needs to be confirmed with further studies, preferably at an individual animal level.

A multi-country One Health foodborne outbreak simulation exercise: cross-sectoral cooperation, data sharing and communication.

Introduction: The awareness of scientists and policy makers regarding the requirement for an integrated One Health (OH) approach in responding to zoonoses has increased in recent years. However, there remains an overall inertia in relation to the implementation of practical cross-sector collaborations. Foodborne outbreaks of zoonotic diseases continue to affect the European population despite stringent regulations, evidencing the requirement for better 'prevent, detect and response' strategies. Response exercises play an essential role in the improvement of crisis management plans, providing the opportunity to test practical intervention methodologies in a controlled environment. Methods: The One Health European Joint Programme simulation exercise (OHEJP SimEx) aimed at practicing the OH capacity and interoperability across public health, animal health and food safety sectors in a challenging outbreak scenario. The OHEJP SimEx was delivered through a sequence of scripts covering the different stages of a Salmonella outbreak investigation at a national level, involving both the human food chain and the raw pet feed industry. Results: A total of 255 participants from 11 European countries (Belgium, Denmark, Estonia, Finland, France, Italy, Norway, Poland, Portugal, Sweden, the Netherlands) took part in national level two-day exercises during 2022. National evaluations identified common recommendations to countries aiming to improve their OH structure to establish formal communication channels between sectors, implement a common data sharing platform, harmonize laboratory procedures, and reinforce inter-laboratory networks within countries. The large proportion of participants (94%) indicated significant interest in pursuing a OH approach and desire to work more closely with other sectors. Discussion: The OHEJP SimEx outcomes will assist policy makers in implementing a harmonized approach to cross-sector health-related topics, by highlighting the benefits of cooperation, identifying gaps in the current strategies and suggesting actions required to better address foodborne outbreaks. Furthermore, we summarize recommendations for future OH simulation exercises, which are essential to continually test, challenge and improve national OH strategies.

Decision-support tools to build climate resilience against emerging infectious diseases in Europe and beyond.

Climate change is one of several drivers of recurrent outbreaks and geographical range expansion of infectious diseases in Europe. We propose a framework for the co-production of policy-relevant indicators and decision-support tools that track past, present, and future climate-induced disease risks across hazard, exposure, and vulnerability domains at the animal, human, and environmental interface. This entails the co-development of early warning and response systems and tools to assess the costs and benefits of climate change adaptation and mitigation measures across sectors, to increase health system resilience at regional and local levels and reveal novel policy entry points and opportunities. Our approach involves multi-level engagement, innovative methodologies, and novel data streams. We take advantage of intelligence generated locally and empirically to quantify effects in areas experiencing rapid urban transformation and heterogeneous climate-induced disease threats. Our goal is to reduce the knowledge-to-action gap by developing an integrated One Health-Climate Risk framework.

A Novel High Discriminatory Protocol for the Detection of Borrelia afzelii, Borrelia burgdorferi Sensu Stricto and Borrelia garinii in Ticks.

Bacteria of the Borrelia burgdorferi sensu lato complex are the causative agents of Lyme borreliosis (LB). Even if the conventional diagnosis of LB does not rely on the species itself, an accurate species identification within the complex will provide a deepened epidemiological scenario, a better diagnosis leading to a more targeted therapeutic approach, as well as promote the general public's awareness. A comparative genomics approach based on the 210 Borrelia spp. genomes available in 2019 were used to set up three species-specific PCR protocols, able to detect and provide species typing of Borrelia afzelii, Borrelia burgdorferi sensu stricto (s.s.) and Borrelia garinii, the three most common and important human pathogenic Lyme Borrelia species in Europe. The species-specificity of these protocols was confirmed on previously identified B. afzelii, B. burgdorferi s.s. and B. garinii specimens detected in Ixodes ricinus samples. In addition, the protocols were validated on 120 DNA samples from ticks collected in Sweden, showing 88% accuracy, 100% precision, 72% sensitivity and 100% specificity. The proposed approach represents an innovative tool in epidemiological studies focused on B. burgdorferi s.l. occurrence in ticks, and future studies could suggest its helpfulness in routine diagnostic tests for health care.

Linking climate and infectious disease trends in the Northern/Arctic Region.

Recognition of climate-sensitive infectious diseases is crucial for mitigating health threats from climate change. Recent studies have reasoned about potential climate sensitivity of diseases in the Northern/Arctic Region, where climate change is particularly pronounced. By linking disease and climate data for this region, we here comprehensively quantify empirical climate-disease relationships. Results show significant relationships of borreliosis, leptospirosis, tick-borne encephalitis (TBE), Puumala virus infection, cryptosporidiosis, and Q fever with climate variables related to temperature and freshwater conditions. These data-driven results are consistent with previous reasoning-based propositions of climate-sensitive infections as increasing threats for humans, with notable exceptions for TBE and leptospirosis. For the latter, the data imply decrease with increasing temperature and precipitation experienced in, and projected for, the Northern/Arctic Region. This study provides significant data-based underpinning for simplified empirical assessments of the risks of several infectious diseases under future climate change.

Screening of Eurasian Tundra Reindeer for Viral Sequences by Next-Generation Sequencing.

Reindeer husbandry is essential for the livelihood and culture of indigenous people in the Arctic. Parts of the herding areas are also used as pastures for farm animals, facilitating potential transmission of viruses between species. Following the Covid-19 pandemic, viruses circulating in the wild are receiving increased attention, since they might pose a potential threat to human health. Climate change will influence the prevalence of infectious diseases of both humans and animals. The aim of this study was to detect known and previously unknown viruses in Eurasian tundra reindeer. In total, 623 nasal and 477 rectal swab samples were collected from reindeer herds in Fennoscandia, Iceland, and Eastern Russia during 2016-2019. Next-generation sequencing analysis and BLAST-homology searches indicated the presence of viruses of domesticated and wild animals, such as bovine viral diarrhea virus, bovine papillomavirus, alcephaline herpesvirus 1 and 2, deer mastadenovirus B, bovine rotavirus, and roe deer picobirnavirus. Several viral species previously found in reindeer and some novel species were detected, although the clinical relevance of these viruses in reindeer is largely unknown. These results indicate that it should be possible to find emerging viruses of relevance for both human and animal health using reindeer as a sentinel species.

First records of adult Hyalomma marginatum and H. rufipes ticks (Acari: Ixodidae) in Sweden.

From July 2018 to January 2019 we recorded 41 specimens of adult Hyalomma ticks, which had been found on horses, cattle or humans in 14 Swedish provinces. In 20 cases we received tick specimens, which were identified morphologically as adults of H. marginatum (n = 11) or H. rufipes (n = 9). These are the first documented records in Sweden of adults of H. marginatum and H. rufipes. Molecular tests for Crimean-Congo haemorrhagic fever virus and piroplasms (Babesia spp. and Theileria spp.) proved negative; 12 out of 20 tested specimens were positive for rickettsiae (R. aeschlimannii was identified in 11 of the ticks). All ticks originated from people or animals that had not been abroad during the previous two months. These data suggest (i) that the adult Hyalomma ticks originated from immature ticks, which had been brought from the south by migratory birds arriving in Sweden during spring or early summer; and that (ii) due to the exceptionally warm summer of 2018 these immature ticks had been able to develop to the adult stage in the summer and/or autumn of the same year. The rapidly changing climate most likely now permits these two Hyalomma species to develop to the adult, reproductive stage in northern Europe. There is consequently a need to revise the risk maps on the potential geographic occurrence of relevant tick species and related tick-borne pathogens in Sweden and in the neighbouring countries.

Discrepancies in data reporting of zoonotic infectious diseases across the Nordic countries - a call for action in the era of climate change.

Emerging infections have in recent years caused enormous health problems. About 70% of these infections are zoonotic e.g. arise from natural foci in the environment. As climate change impacts ecosystems there is an ongoing transition of infectious diseases in humans. With the fastest changes of the climate occurring in the Arctic, this area is important to monitor for infections with potentials to be climate sensitive. To meet the increasing demand for evidence-based policies regarding climate-sensitive infectious diseases, epidemiological studies are vital. A review of registered data for nine potentially climate-sensitive infections, collected from health authorities in Denmark/Greenland, Finland, Iceland, Norway and Sweden, found that performing such studies across countries is constrained by incompatible reporting systems and differences in regulations. To address this, international standardisation is recommended.

Identifying climate-sensitive infectious diseases in animals and humans in Northern regions.

BACKGROUND: General knowledge on climate change effects and adaptation strategies has increased significantly in recent years. However, there is still a substantial information gap regarding the influence of climate change on infectious diseases and how these diseases should be identified. From a One Health perspective, zoonotic infections are of particular concern. The climate in Northern regions is changing faster than the global average. This study sought to identify climate-sensitive infectious diseases (CSIs) of relevance for humans and/or animals living in Northern regions. Inclusion criteria for CSIs were constructed using expert assessments. Based on these principles, 37 potential CSIs relevant for Northern regions were identified. A systematic literature search was performed in three databases using an explicit stepwise approach to determine whether the literature supports selection of these 37 potential CSIs. RESULTS: In total, 1275 nominated abstracts were read and categorised using predefined criteria. Results showed that arthropod vector-borne diseases in particular are recognised as having potential to expand their distribution towards Northern latitudes and that tick-borne encephalitis and borreliosis, midge-borne bluetongue and the parasitic infection fasciolosis can be classified as climate-sensitive. Many of the other potential CSIs considered are affected by extreme weather events, but could not be clearly classified as climate-sensitive. An additional literature search comparing awareness of climate influences on potential CSIs between 1997-2006 and 2007-2016 showed an increase in the number of papers mentioning effects of climate change. CONCLUSIONS: The four CSIs identified in this study could be targeted in a systematic surveillance programme in Northern regions. It is evident that climate change can affect the epidemiology and geographical range of many infectious diseases, but there were difficulties in identifying additional CSIs, most likely because other factors may be of equal or greater importance. However, climate-ecological dynamics are constantly under change, and therefore diseases may fall in or out of the climate-sensitive definition over time. There is increasing awareness in the literature of the effects of climate change on infectious diseases over time.

Seroprevalence of pestivirus in Eurasian tundra reindeer in Finland, Sweden, Norway, Iceland and Russian Federation.

Reindeer herding is of great importance for the indigenous people of the Fennoscandia peninsula and northern Russia. There are also free-ranging feral populations of reindeer in Finland, Iceland, Norway and Russian Federation. The genus Pestivirus contains several viral species that infect ungulates and often show capacity to transmit between different host species. Sera from 520 Eurasian tundra reindeer (Rangifer tarandus tarandus) from Finland, Sweden, Norway, Iceland and Russian Federation were analysed and the prevalence of pestivirus-specific antibodies was determined. Seropositivity proportion was 48.5% for Sweden and 41.2% for Norway, but only 1.6% for Iceland and 2.5% for Finland. All Russian reindeer investigated were seronegative. Pan-pestivirus RT-PCR of seronegative animals (n = 156) from seropositive herds confirmed their negative status. These results indicate unexpectedly non-uniform circulation of an as yet uncharacterised pestivirus in Eurasian reindeer populations. The high seroprevalence in some regions warrants further studies of pestivirus infection dynamics, effects on reindeer health and population dynamics.